The expression of the HSV-1 glycoprotein C (gC) gene was studied by using a chimeric gene that was constructed by cloning the promoter sequences of the gC gene upstream of the coding sequences for the bacterial enzyme Beta-galactosidase (Beta-gal), such that expression of Beta-gal was directed by the gC regulatory region. This chimeric gene was cloned as a unit into the middle of the coding sequences of the HSV-1 thymidine kinase (TK) gene and, by homologous recombination, was inserted into the HSV-1 genome at the TK locus. A recombinant virus containing the chimeric gene was isolated by screening for TK virus that expressed Beta-gal, and this virus was shown to express Beta-gal as a late HSV-1 gene. To define the sequences necessary for viral late gene regulation, a series of deletions was made from the 5' end of the 1450 base-pair gC regulatory region and the effect of the deletions on Beta-gal expression was examined both in transient assays and in recombinant viruses. In both types of assay, deletions which extended to -104 bp above the mRNA start site has no effect on Beta-gal expression, indicating that late gene regulatory signals in the 5' region of the gC gene lie within this 104 bp. Other deletions have been made in both the 5' and the 3' regions of the gC regulatory sequences and are being used to further define the elements of regulatory control for the gC gene.